The present invention relates generally to CNS cell lines. The invention is more particularly related to conditionally-immortalized human CNS progenitor cell lines and to differentiated cells derived from such cell lines. Such cell lines and/or differentiated cells may be used in the development of therapeutic agents for the prevention and treatment of neurological diseases and other conditions. The present invention is also related to the use of such cell lines and/or differentiated cells in assays and for the study of CNS cell development, death and abnormalities.
The development of therapies for central nervous system (CNS) disorders has been hampered by the lack of human cells for research and development. Human CNS tissue is difficult to obtain and is not available on a regular basis. Primary human CNS cultures derived from such tissue generally express neuronal markers and contain functional ion channels and neurotransmitter receptors, but such cultures have a limited life span (about one month), necessitating frequent dissections and plating. Accordingly, neither tissue nor primary cultures are capable of supplying the cells needed for extensive research and development.
Drug development is generally carried out using immortalized rodent cells, and the results are then extrapolated to humans. Rodent neuronal progenitor cells have been immortalized with, for example, retroviral vectors encoding the myc oncogene or SV40 large T antigen (Bartlett et al., Proc. Natl. Acad. Sci. USA 85:3255-3259, 1988); Cepko, Ann. Rev. Neurosci. 12:47-65, 1989; Eves et al., Proc. Natl. Acad. Sci. USA 89:4373-4377, 1992; Frederiksen et al., Neuron 1:439-448, 1988; Gage et al., Ann. Rev. Neurosci. 18:159-192, 1995; Gao and Hatten, Development 120:1059-1070, 1994; Giordano et al., Exp. Neurol. 124:395-400, 1993; Lendhal and McKay, TINS 13:132-137, 1990; Mehler et al., Nature 362:62-65, 1993; Renfranz et al., Cell 66:713-729, 1988; Ryder et al., J. Neurobio. 21:356-375, 1990; White et al., J. Neurosci. 14:6744-6753, 1994; Whittemore and White, Brain Res. 615:27-40, 1993). However, in v-myc immortalized cells, the mitotic activity of the oncogene is always present; cells do not undergo differentiation, and channels and receptors are not functional. To allow differentiation, a temperature-sensitive mutant of SV40 large T-antigen (tsA58) has been used (Eves et al., Proc. Natl. Acad Sci. USA 89:4373-4377, 1992; Mehler et al., Nature 362:62-65, 1993). At the non-permissive temperature (39xc2x0 C.) the expression of T-antigen in transfected cells is considerably down-regulated. However, the progenitor cells can undergo only incomplete differentiation into neurons (Frederiksen et al., Neuron 1:439-448, 1988; Gao and Hatten; Development 120:1059-1070, 1994; Mehler et al., Nature 362:62-65, 1993; Renfranz et al., Cell 66:713-729, 1988; White et al., J. Neurosci. 14:6744-6753, 1994; Whittemore and White, Brain Res. 615:27-40, 1993). A combination of factors and substrates is needed to differentiate the cells further in vitro, and complete differentiation of these cells has not been achieved. In addition, species differences continue to render the use of rat CNS cells problematic. In particular, the small variations in specific protein sequences between species may translate into significant effects when pharmacological agonists or antagonists are examined.
Cloned human CNS channels and receptors provide a potential solution to this problem, but such cloned proteins are not present in their native environment (typically they are expressed in cell lines derived from non-CNS tissue, such as kidney (HEK 293) or cervical (HeLa) cells) and downstream signaling pathways are abnormal. Moreover, these receptors are heteromultimeric proteins and, while the exact stoichiometry of subunits is not known, it is likely that there are numerous subtypes with different subunit compositions. Expressed receptor subunit combinations are artificial and may not reflect those in vivo, and attempts to match native receptors with heterologously expressed subunits have not been successful in a number of cases. Consequently, expressed channels and receptors differ fundamentally from their native counterparts and are not optimal for drug development.
Human CNS lines containing functional native channels and receptors in their normal cellular environment would provide an infinite and homogeneous source of human CNS cells and would offer significant advantages for CNS drug discovery. Currently, the only human cell line that can be differentiated into postmitotic CNS neurons is a human teratocarcinoma cell line (NT2) that requires 6-10 weeks of complex in vitro manipulations for neuronal differentiation (Younkin et al., Proc. Natl. Acad. Sci. USA 90:2174-2178, 1993). This lengthy time period is inconvenient for basic research and prohibitive for use in high-throughput screens of thousands of compounds. Human CNS lines that can be differentiated in a shorter period of time into neurons expressing functional receptors would clearly be preferable. Thus far, however, it has not been possible to generate such cell lines, and the techniques for generating the rat progenitor cell lines have been largely unsuccessful when applied to human cells.
Accordingly, there is a need in the art for human CNS lines that may be readily differentiated and that express channels and receptors in their native forms. The present invention fulfills these needs and further provides other related advantages.
Briefly stated, the present invention provides conditionally-immortalized human CNS progenitor cell lines capable of differentiation into astrocytes and/or neurons. In one aspect, the present invention provides a method for producing a conditionally-immortalized human CNS progenitor cell, comprising (a) plating human progenitor cells on a surface that permits proliferation; (b) adding growth medium to the cells; (c) transfecting the cells with DNA encoding a selectable marker and regulatable growth-promoting gene under conditions promoting expression of the growth-promoting gene; (d) passaging the transfected cells onto a substrate; and (e) adding growth medium supplemented with one or more proliferation-enhancing factors to the transfected cells.
In a related aspect, the present invention provides a conditionally-immortalized clonal human CNS progenitor cell capable of differentiation into neurons and astrocytes.
In another aspect, a method for producing astrocytes and/or neurons is provided comprising culturing a cell produced as described above under conditions inhibiting expression of the growth-promoting gene. In a related aspect, astrocytes and neurons prepared as described above are provided.
In yet another aspect, the present invention provides a method for introducing a CNS cell into a mammal, comprising administering to a mammal a cell as described above.
In a related aspect, a method for treating a patient is provided, comprising administering to a patient a cell as described above.
In a further aspect, the present invention provides methods for screening for an agent that modulates activity of a protein produced by a CNS cell, comprising (a) contacting a cell as described above and (b) subsequently measuring the ability of said candidate agent to modulate activity of a protein produced by said cell.
In yet another aspect, a method for detecting the presence or absence of a protein in a sample is provided comprising (a) contacting a sample with a cell as described above and (b) subsequently detecting a response in said cell, and thereby detecting the presence of a protein in said sample.
In a further aspect, the present invention provides a method for identifying a human CNS gene or protein, comprising detecting the presence of a gene or protein within a culture of cells as described above.
In another aspect, a method is provided for screening for an agent that affects CNS cell death, comprising (a) contacting a cell as described above with a candidate agent under conditions that, in the absence of candidate agent, result in death of said cell and (b) subsequently measuring the ability of said candidate agent to affect the death of said cell.
In a related aspect, the present invention provides a method for screening for a protein that regulates CNS cell death, comprising (a) altering the level of expression of a protein within a cell as described above and (b) subsequently measuring the affect of said alteration on the death of said cell, and thereby identifying a protein that regulates CNS cell death.
In a further aspect, conditionally-immortalized human CNS progenitor cells are provided.